Bone remodeling is a continuously occurring balance of bone destruction followed by bone formation, which maintains bone strength by replacing damaged bone with new bone. Unfortunately, diseases affecting the skeleton such as osteoporosis, rheumatoid arthritis, and cancer, tilt the delicate balance of bone destruction and bone formation toward the prior, leading to excessive bone loss. In many cases, this bone loss causes significant morbidity including severe pain, joint destruction, and pathological fracture. Despite advances in our comprehension of the bone remodeling process, the mechanisms responsible for pathological bone loss initiated by skeletal diseases such as osteoporosis, rheumatoid arthritis, and cancer remain poorly understood. Improved understanding of these mechanisms is vital to the development of more effective therapeutics. Recent evidence suggests that members of the chemokine family of chemical messengers may play a role in mediating pathological bone loss. The chemokine macrophage inflammatory protein (MIP)-3a, whose expression has been reported in both rheumatoid arthritis and periodontitis, has been shown to enhance the development of bone-destroying cells called osteoclasts (OCL). Interestingly, we recently found levels of the MIP family member MIP-1d to be significantly elevated in renal cell carcinoma bone metastasis (RBM), which frequently causes excessive bone destruction. Interestingly, MIP-1d has also been detected in synovial tissue from rheumatoid arthritis patients. Subsequent preliminary studies in vitro demonstrated that MIP-1d not only stimulates recruitment of immature OCL, but also enhances differentiation into mature OCL. Based on these preliminary data, we hypothesize that MIP-1d plays a direct role in mediating pathological bone loss by enhancing stimulation of key signaling pathways of OCL formation in combination with RANKL. In preliminary studies we found that both RANKL and MIP-1d were capable of stimulating NF-?B and PLC?2 signaling, two critical pathways for OCL formation, while other important pathways in OCL formation were unaffected by MIP-1d. We will initially investigate our hypothesis by determining the ability of MIP-1d to enhance NF-?B and PLC?2 activation in combination with RANKL. Subsequently, we will utilize chemical inhibitors of NF-?B and PLC?2 to examine the functional role of each pathway in MIP-1d-enhanced OCL formation in vitro. Lastly, using human RBM cell lines in which MIP-1d production has been increased or silenced, we will examine the effect of MIP-1d on bone destruction in vivo using a mouse model of RBM- induced bone loss. In addition, we will determine the clinical correlation between MIP-1d expression in primary RCC and the subsequent development of osteolytic metastasis. These studies will further our knowledge regarding the role of chemokines in pathological bone loss and may provide a novel target for prevention and therapy, thus having the potential to greatly impact the future of Orthopaedic medicine. PUBLIC HEALTH RELEVANCE: This work may identify MIP-1d as a therapeutic target for the prevention and treatment of pathological bone loss, provide insight into how the effects of MIP-1d may be abrogated, and determine the potential utility of MIP-1d as a prognostic indicator for both the development of osteolytic metastasis and the therapeutic response to anti- RANKL therapy, which is currently in phase 3 studies evaluating its effects in patients with conditions resulting in bone loss (eg. osteoporosis, rheumatoid arthritis, bone metastasis, etc.). Therefore, this work is timely and has the potential to influence both clinical prevention and treatment decisions, providing the framework necessary for future pre-clinical and clinical studies, and will greatly advance our current understanding of the biological mechanisms involved in the development of pathological bone loss in diseases such as rheumatoid arthritis and cancer, which affect millions of people each year. If more effective medical therapy can be instituted prior to or early in the course of disease, the development of severe bone destruction may be prevented or delayed, resulting in improved quality of life and decreased costs of care for these patients who often require surgical treatment, thus greatly impacting public health.